Diagnostic value for methylation in cervical cancer based on a small‐molecule fluorescent probe targeting DNMT1

Abstract Background Value analysis of a small‐molecule fluorescent probe for methylation detection in different cervical lesions. Materials and Methods (1) The grayscale values of distinct lesion tissues were remarkably distinct among the four groups (p < 0.05). The comparison of the grayscale value between the two groups showed that the CA group noticeably exceeded the LSIL and cervicitis groups, and the HSIL group was apparently higher than the LSIL and cervicitis groups (p < 0.05); (2) The mean grayscale values of the enrolled subjects were calculated with 55.21 as the midline, with >55.21 as positive and ≤55.21 as negative. Results The results showed that the positive rate of the cervicitis group was 0.00%, the LSIL group 67.74%, the HSIL group 83.33%, and the CA group 100.00%. The results among the four groups were notably distinct (p < 0.05); (3) The comparison among DAPI, probe, bright, and merged images of cervicitis, LSIL, HSIL, and CA indicated that different cervical lesions were with quite various stains. Conclusion The grayscale value, positive rate, and stained picture of distinct cervical lesions were remarkably different. The small‐molecule fluorescent probe has a good value in differentiating cervical lesions and can be considered for popularization and application.

persistent infection cannot be distinguished, while women of reproductive age have a higher HPV infection rate, suggesting that HPV screening alone is of weak significance. 7,8The application of molecular biology detection in the screening of cervical cancer has been popularized.

General data
Cervical tissues from 81 patients who visited our cervical specialist clinic from August 2020 to January 2022 were retrospectively registered, and the enrolled tissues were divided into cervicitis group

Quantitative detection of DNMT1 by a small-molecule fluorescent probe in cervical cell
PAX1 methylation is primarily mediated by DNA methyltransferase 1 (DNMT1), and DNMT1 plays an important role as a PAX1 methylation promoter in cervical cancer cells.Our team, in collaboration with the Pharmaceutical Chemistry Laboratory of Fudan University, designed and synthesized a small fluorescent probe RG108-FITC (patent number: CN201910750081.X), deriving from the combination of ligands or inhibitors of DNMT1 with the specific fluorescent group.DNMT1 level in cells can be measured via the probe by specifically binding to DNMT1, so indirectly evaluating the methylation level of PAX1.
For quantitative DNMT1 fluorescence assays, the cells were diluted with medium, counted, and transferred to a new centrifuge tube after centrifuging the cell suspension, and then an appropriate volume of medium containing the small-molecule fluorescent probe was added until the volume was up to 1 mL.After incubation at 37 • C for 4 h, the cells were harvested and treated with DAPI (0.1 µg/mL) for 10 min to stain cell nuclei.Images of DAPI and fluorescent probes were obtained by confocal fluorescence microscopy (Nikon) at 349 and 488 nm, respectively.Fluorescence intensity was calculated by ImageJ.

Outcome measurements
The primary outcome was the efficacy of the probe in differentiating groups was carried out.Finally, the stained pictures of typical cases in the cervicitis, LSIL, HSIL, and CA groups were screened for comparison.

Statistical methods
The results were expressed as mean ± standard deviation.Data that did not conform to a normal distribution were statistically calculated using the Kruskal Wallis rank sum test, and the results were expressed as median and quartiles.Enumeration data were expressed as rates, and statistics were performed by chi-square test.Differences were considered significant at p < 0.05.GraphPad Prism 8.3 was conducted for plotting.

Comparison of the greyscale values of the small-molecule probe in distinct cervical lesion cell
A comparison of the greyscale values of the small-molecule probe in distinct lesion tissues suggested that the difference was remarkable among all groups (p < 0.001).Comparison between the two groups revealed that the greyscale value in the CA group was noticeably higher than that in the LSIL and cervicitis groups (p < 0.05), and in the HSIL group, the greyscale values considerably exceeded that of the LSIL and cervicitis groups (p < 0.05).Table 1 shows a comparison of small-molecule probe greyscale values for distinct cervical lesion tissues, Figure 1 shows a comparison of the greyscale values of the small-molecule probe in distinct cervical lesion tissues.

Evaluate the positive rate of the grayscale values in distinct lesion tissues
Taking the mean grayscale value of 55.21 as the midline, >55.21 as the positive, and ≤55.21 as the negative, the positive rates of the grayscale values of distinct lesion tissues were calculated, respectively.
The results suggested that the positive rate of the cervicitis group was 0.00%, the LSIL group 67.74%, the HSIL group 83.33%, and the CA group 100.00%.The positive rate among the four groups was strikingly F I G U R E 1 Comparison of the greyscale values of the small-molecule probe in distinct cervical lesion tissues.The greyscale values were strikingly higher in the CA group in contrast to the LSIL and cervicitis groups, and the HSIL group was found a notable increase compared with the LSIL and cervicitis groups (both p < 0.05).

TA B L E 2
Positive rate of the greyscale values in distinct cervical lesions.The positive rates of the greyscale in distinct cervical tissues were significantly different (p < 0.05).Further two-by-two comparison showed that the positive rate in the CA group was higher than that in the cervicitis, LSIL, and HSIL, the HSIL group higher than the cervicitis group, and the LSIL group higher than the cervicitis group (p < 0.05).

Group
distinct (p < 0.05).Further pairwise comparison indicated that the positive rate of the CA group was superior to that of the cervicitis group, LSIL, and HSIL group, the HSIL group higher than the cervicitis group, and the LSIL group higher than the cervicitis group (all p < 0.05), Table 2 shows positive rate of the greyscale values in distinct cervical lesions.
Figure 2 shows the difference in the greyscale values of distinct lesions.

Stained images of typical cases
With DAPI images, probe images, bright images, and merged images of patients with cervicitis, LSIL, HSIL, and CA screened for Comparison, it is more evident that the stained images of patients with distinct cervical lesions varied greatly.Figure 3 shows stained images of patients with cervicitis, and Figure 4 shows stained images of patients with LSIL.
Figure 5 shows Stained images of patients with HSIL; Figure 6 shows stained images of patients with cervical cancer.

DISCUSSION
In recent years, some areas have shown an obvious tendency to rejuvenation for cervical cancer, with an average age of 52 years of carcinoma onset in situ having subsided to 30-34 years, nearly 20 years earlier than invasive cancer. 9,10In China, the age of onset of cervical cancer has also gradually decreased in recent years. 11,12130 000 new cases, and 53 000 deaths due to cervical cancer in China each year, accounting for about 18% of cervical cancer deaths worldwide. 13,14thylation is a type of chemical modification of DNA, which refers to the process by which methyltransferases covalently bind the methyl group of S-adenosylmethionine to the cytosine 5′ carbon of CpG dinucleotides in the DNA sequence without changing the DNA sequence.
Methylation modification of cytosine in DNA plays a crucial role in gene regulation to cell growth and development. 15,16Methylation is not only a vital element in maintaining normal biological functions, but is closely related to the occurrence and development of many diseases, such as extensive hypomethylation in tumors, massive demethylation of retrotransposons, and hypermethylation of specific CpG islands.It can be said that aberrant methylation of DNA is an early phase in the process of tumorigenesis, and early screening for methylated genes will help provide new ideas for the diagnosis and healing of tumors. 17,18ne epigenetic modification is the main feature of cervical can-  Although there are differences in the methylation time of different genes, the methylation level will increase with the process of carcinogenesis overall which is correlated with the severity of cervical diseases.Therefore, the methylation status of genes can be used as an evaluation of the degree of cervical lesions. 22,23A comparison of stained images proved that a significant variety existed in distinct cervical lesions.Although the judgment of stained degree was affected by individual subjective consciousness and detection instruments, the result also confirmed that the methylation level was closely related to the extent of cervical lesions.
The study demonstrated that the innovation of a small-molecule fluorescent probe based on gene methylation detection was of high application value in terms of quantitative, qualitative, and picture visualization.The limitation was that the samples came from a single and concentrated source, which may impact the results to a certain extent.

CONCLUSION
The grayscale value, positive rate, and stained picture of distinct cervical lesions were remarkably distinct.The small-molecule fluorescent probe based on gene detection technology owned a promising prospect in the identification of cervical lesions and could be considered for popularization and application.

F I G U R E 2
,c Represent p < 0.05, contrasted to the cervicitis, the LSIL, and the HSIL group, respectively.Difference in the greyscale values of distinct lesions.
cer, and a large number of genes such as shooting cell cycle regulation, apoptosis, DNA repair, and WNT pathway have been confirmed to have methylation modification phenomena, and studies have shown that the activity of DNA de novo methylase DNMTs is directly increased in patients with high-risk HPV infection, which confirms the correlation between methylation and the pathological process of cervical cancer.[19][20][21]In the present paper, 81 patients with distinct cervical lesions were examined by a gene detection technique via a smallmolecule fluorescent probe to explore the application value of this method in distinguishing different cervical lesion types.The results showed that there were remarkable differences in the cervicitis, LSIL, HSIL, and cervical cancer patients in terms of quantitative data of the grayscale values.Further comparison of the positive rates according to the mean values showed that there were also noticeable differences among distinct cervical lesion tissues, and the pairwise comparison also had certain statistical significance.Previous in vitro experiments have confirmed that the degree of methylation is associated with the degree of cervical lesions and the course of high-grade endocervical neoplasia during carcinogenesis.

F I G U R E 3 F I G U R E 4 F I G U R E 5
Stained images of patients with cervicitis.(A) DAPI image, (B) probe image, (C) bright image, and (D) merged image.Stained images of patients with LSIL.(A) DAPI image, (B) probe image, (C) bright image, and (D) merged image.Stained images of patients with HSIL.(A) DAPI image, (B) probe image, (C) bright image, and (D) merged image.F I G U R E 6 Stained images of patients with cervical cancer.(A) DAPI image, (B) probe image, (C) bright image, and (D) merged image.